Inclusion of pyridoxine dependent epilepsy in expanded newborn screening programs by tandem mass spectrometry: set up of first and second tier tests

Pyridoxine-dependent epilepsy (PDE) is a rare genetic disorder characterized by intractable neonatal seizures responsive to pyridoxine. Diagnosis relies on quantification of α-aminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid in urine or plasma in patients with overt symptoms. We developed and validated simple and rapid first- and second-tier methods for two recently published biomarkers of PDE (2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) and 6-oxopiperidine-2-carboxylic acid (6-oxoPIP)) in extended newborn screening (NBS) programs from neonatal dried blood spots (DBS). For the first-line test, DBS specimens were collected from 5,405 newborns who underwent routine NBS and analysed by FIA-MS/MS. For the second-tier test, samples were analysed by LC-MS/MS. The neonatal DBS from two patients with genetically confirmed PDE were also analysed. The reference values for NBS resulted <0.34 μmol/L for 2-OPP and <4.51 μmol/L for 6-oxoPIP. In the second-tier test, limits of detection were 0.07 μmol/L and 0.14 μmol/L, whereas limits of quantification were 0.25 μmol/L and 0.48 μmol/L, respectively, for 2-OPP and for 6-oxoPIP. The tests provided good linearity, reproducibility, accuracy and precision, with acceptable matrix effect and carry-over, according to international validation criteria. The biomarkers in DBS were stable at room temperature, +4 °C and -20 °C for one month. When assessing these biomarkers in two patients with genetically confirmed PDE, the higher sensitivity of 2-OPP as compared to 6-oxoPIP in discriminating PDE emerged. The first-line and second-tier tests developed in this study highlight the potential for including PDE in the NBS panel, early diagnosis and prompt precision treatment initiation.